The present invention relates generally to a family of novel protein factors which share homologous sequences and biological activities with megakaryocyte colony-stimulating factor (meg-CSF) and which participate in the differentiation or maturation of megakaryocyte progenitors. Also provided are processes for obtaining and producing the factors from natural sources or by artificial means, e.g., chemical synthesis or recombinant genetic engineering techniques.
Megakaryocytes are the hematopoietic cells, largely found in the bone marrow, but also in peripheral blood and perhaps other tissues as well, which produce platelets (also known as thrombocytes) and subsequently release them into circulation. Megakaryocytes, like all of the hematopoietic cells of the human hematopoietic system, ultimately are derived from a primitive stem cell after passing through a complex pathway comprising many cellular divisions and considerable differentiation and maturation.
The platelets derived from these megakaryocytic cells are critical for maintaining hemostasis and for initiating blood clot formation at sites of injury. Platelets also release growth factors at the site of clot formation that speed the process of wound healing and may serve other functions. However, in patients suffering from depressed levels of platelets (thrombocytopenia) the inability to form clots is the most immediate and serious consequence, a potentially fatal complication of many therapies for cancer. Such cancer patients are generally treated for this problem with platelet transfusions. Other patients frequently requiring platelet transfusions are those undergoing bone marrow transplantation or patients with aplastic anemia.
Platelets for such procedures are obtained by plateletphoresis from normal donors. Like most human blood products, platelets for transfusion have a relatively short shelf-life and also expose the patients to considerable risk of exposure to dangerous viruses, such as the human immunodeficiency virus (HIV) or a hepatitis virus.
Clearly the ability to stimulate endogenous platelet formation in thrombocytopenic patients with a concomitant reduction in their dependence on platelet transfusion would be of great benefit. In addition, the ability to correct or prevent thrombocytopenia in patients undergoing radiation therapy or chemotherapy for cancer would make such treatments safer and possibly permit increases in the intensity of the therapy thereby yielding greater anti-cancer effects.
For these reasons considerable research has been devoted to the identification and purification of factors involved in the regulation of megakaryocyte and platelet production. Although there is considerable controversy on this subject, the factors regulating the growth and differentiation of hematopoietic cells into mature megakaryocytes and the subsequent production of platelets by these cells are believed to fall into two classes: (1) megakaryocyte colony-stimulating factors (meg-CSFs) which support the proliferation and differentiation of megakaryocytic progenitors in culture, and (2) thrombopoietic (TPO) factors which support the differentiation and maturation of megakaryocytes in vivo, resulting in the production and release of platelets. [See, e.g., E. Mazur, Exp. Hematol., 15:340-350 (1987).]
Each class of factors can be defined by bioassay. Factors with meg-CSF activity support megakaryocyte colony formation, while factors with TPO activity elicit an elevation in the numbers of circulating platelets when administered to animals. It is not clear how many species of factors exist that have either one or both of these activities. For example, human IL-3 supports human megakaryocyte colony formation and, at least in monkeys, frequently elicits an elevation in platelet count. However, IL-3 influences hematopoietic cell development in all of the hematopoietic lineages and can be distinguished from specific regulators of megakaryocytopoiesis and platelet formation which interact selectively with cells of the megakaryocytic lineage.
Many different reports in the literature describe factors which interact with cells of the megakaryocytic lineage. Several putative meg-CSF compositions have been derived from serum [See, e.g., R. Hoffman et al, J. Clin. Invest., 75:1174-1182 (1985); J. E. Straneva et al, Exp. Hematol., 15:657-663 (1987); E. Mazur et al, Exp. Hematol., 13:1164-1172 (1985]. A large number of reports of a TPO factor are in the art. [See, e.g., T. P. McDonald, Exp. Hematol., 16:201-205 (1988); T. P. McDonald et al, Biochem. Med. Metab. Biol., 37:335-343 (1987); T. Tayrien et al, J. Biol. Chem., 262: 3262-3268 (1987) and others]. From the studies reported to date, it is not clear whether factors having activities identified as meg-CSF also have TPO activity or vice versa.
Although there have been numerous additional reports tentatively identifying these regulatory factors, the biochemical and biological identification and characterization of meg-CSF and TPO factors have been hampered by the small quantities of the naturally occurring factors which are present in natural sources, e.g., blood and urine.
The present inventors have previously identified a purified meg-CSF factor from urine described in PCT application No. WO91/02001, published Feb. 21, 1991, and further described in pending U.S. patent application Ser. No. 07/643,502 now abandoned. This homogeneous meg-CSF, purified from urine and which may be produced via recombinant or synthetic techniques, is characterized by a specific activity in the murine fibrin clot assay of greater than 5xc3x97107 dilution units per mg and preferably, 2xc3x97108 dilution units per mg protein. This meg-CSF was processed from a precursor protein encoded by an approximately 18.2 kb genomic clone which contains ten exons. Two additional exons were subsequently identified outside this 18.2 kb segment. cDNAs made from the full length cDNA and from partial cDNAs have been expressed in COS-1 cells and CHO cells. These references are incorporated herein by reference for the disclosure of that meg-CSF molecule.
There remains a need in the art for additional proteins either isolated from association with other proteins or substances from their natural sources or otherwise produced in homogeneous form, which are capable of stimulating or enhancing the production of platelets in vivo, to replace presently employed platelet transfusions and to stimulate the production of other cells of the lymphohematopoietic system.
In one aspect the present invention provides a family of human megakaryocyte stimulating factors (hereafter called MSFs) or biologically active peptide fragments thereof that are substantially free from association with proteins or substances with which they occur in nature. The MSF family includes non-naturally occurring MSF proteins which are characterized by sharing similar structure, DNA and amino acid sequences, and!or biological activity with naturally-occurring MSFS. Naturally-occurring MSFs may be isolated and purified from natural sources. Non-naturally occurring MSFs may be prepared by chemical synthesis and/or recombinant genetic engineering techniques.
These MSF proteins comprise DNA sequences encoding one or more of the exon sequences described below in FIG. 1 or biologically active fragments thereof. Each MSF protein of this invention is also characterized by one or a combination of the physical, biochemical, pharmacological or biological activities described herein.
Another aspect of the present invention includes a mixture of different MSFs, and active fragments thereof.
Another aspect of the present invention includes DNA sequences that encode the expression of these naturally occurring and non-naturally occurring MSFs.
Still another aspect of the present invention are recombinant DNA molecules comprising vector DNAs and DNA sequences encoding the MSFs of this invention. Each DNA molecule provides the MSF DNA in operative association with a regulatory sequence capable of directing the replication and expression of each MSF in a selected host cell. Host cells transformed with such DNA molecules for use in expressing recombinant MSF proteins are also provided by the present invention.
The DNA molecules and transformed cells of the invention may be employed in a novel process for producing recombinant MSFs, or biologically active peptide fragments thereof. A cell line, transformed with a DNA sequence encoding expression of individual MSFs or fragments thereof (or a recombinant DNA molecule as described above) in operative association with a suitable regulatory or expression control sequence capable of controlling expression of the protein, is cultured under appropriate conditions permitting expression of the recombinant DNA. The expressed MSF protein is then harvested from the host cell, cell lysate or culture medium by suitable conventional means.
Still a further aspect of the present invention is a process for isolating and purifying an MSF composition of the present invention or a fragment thereof, or a mixture of MSFs and fragments, from natural sources, e.g. urine or peripheral blood leukocytes. Alternatively, the MSFs may be isolated and purified from conditioned medium or cell lysate of cells expressing recombinant MSF protein.
Another aspect of this invention provides pharmaceutical compositions containing a therapeutically effective amount of one or more MSFs of the present invention. These pharmaceutical compositions may be employed in methods for treating disease states or disorders, for example, diseases characterized by a deficiency of platelets and other disorders referred to herein.
A further aspect of the invention, therefore, is a method for treating such disease states by administering to a patient a therapeutically effective amount of at least one MSF in a suitable pharmaceutical carrier. Alternatively, several MSFs may be administered in combination. These therapeutic methods may include administering in combination, simultaneously, or sequentially with an MSF, an effective amount of at least one other TPO-like factor, meg-CSF or other cytokine, e.g, IL-3 or steel factor; hematopoietin, interleukin, growth factor, or antibody.
Still another aspect of the present invention provides antibodies directed against an MSF of this invention. As part of this aspect, therefore, the invention includes cell lines capable of secreting such antibodies and methods for their production and use in diagnostic or therapeutic procedures.
Other aspects and advantages of the present invention will be apparent upon consideration of the following detailed description of preferred embodiments thereof.